The present invention is based, in part, on conventional knowledge of antibodies, the antigens which bind them, and known procedures for making large mixtures of peptides and other molecules which may bind to naturally occurring antibodies. Antibodies are proteins produced by lymphoid cells (plasma cells). The antibodies are produced by the cells in response to "foreign" substances (antigens), either exogenous or endogenous, and are capable of binding specifically with the antigen which stimulated the immune response. The antibodies can also bind with substances which are structurally similar to that same antigen. An antigen can be any foreign substance that, upon introduction into a vertebrate animal, stimulates the production of antibodies. An antigen may also be of endogenous source, such as antigens in auto-immune diseases or from tissue destruction. A complex antigen-like molecule may carry several antigenically distinct sites which are referred to as determinants. A substance which is structurally similar to certain parts of an immunogen (in general the determinants) can react specifically with its antibody. However, such smaller substances are generally too small to stimulate antibody synthesis by themselves and can be referred to as an incomplete antigen or hapten.
Throughout the life of vertebrate animals, such as humans, foreign substances are continually introduced. These foreign substances include viruses and bacteria which cause disease. Each time an antigenic substance is introduced it may result in the generation of a different antibody or several antibodies. Since many foreign substances are introduced over the life of the animal, and since many antibodies may be generated in response to a single foreign substance, each individual animal will have a large numbers of different antibodies. With present technology, it is very difficult to isolate and recover a specific antibody associated with a specific antigen or disease especially if nothing is known about the characteristics of the antibody or antigens involved. More specifically, if one or more individuals is known to have a given disease (wherein the agent responsible for the disease has not been characterized), it is not a simple matter to isolate the antibodies in that individual which have been generated and amplified as a result of that disease, nor is it a simple matter to isolate and recover antigens which bind to those antibodies. The present invention endeavors to provide approaches which make it possible to isolate and recover antibodies which are characteristic of a specific disease and to simultaneously isolate and recover antigens and/or related molecules which specifically bind to those antibodies.
In order to carry out the methodology of the present invention it is necessary to produce large numbers of peptides and/or other molecules which can be tested for their ability to bind to antibodies. These large mixtures of peptides and/or other molecules can be produced using technology described in the literature. One of the initial methods of producing multiple peptides more rapidly than the standard Merrifield method is disclosed by Houghten, R. A., Proc Natl Acad Sci USA (1985) 82:5131-5135. The Houghten method involves a modification of the Merrifield method but uses many individual polyethylene bags resulting in a method wherein each bag will contain a different peptide. An alternative method was devised by Geysen, H. M., et al., Proc Natl Acad Sci USA (1984) 81:3998-4002. (See also, W086/06487 and W086/00991). In accordance with the Geysen method, C-terminal amino acid residues are bound to solid supports in the form of multiple polyethylene pins and the pins treated in parallel to attach additional amino acid residues.
More recently, machines have been introduced which produce many peptides by parallel synthesis (e.g. Advanced Chem Tech, Gilson). An advancement in the ability to produce extremely large numbers of peptides as mixtures was disclosed within U.S. Pat. No. 5,010,175 issued Apr. 23, 1991 to Rutter and Santi. The Rutter and Santi method makes it possible to generate large numbers of peptides in either equimolar amounts or in predictable amounts. The methodology makes it possible to quickly synthesize large numbers of peptides such as mixtures containing 64 million or more different and distinct peptides. Another method for producing mixtures of peptides including large numbers of different peptides is disclosed within issued U.S. Pat. No. 5,182,366 issued Jan. 26, 1993 to Huebner et al. and in publications by Lam et al., Nature, (1991) 354, 82-84, and Prague paper.
The procedures described in the patents listed above allow for the production of peptides and/or modified peptides using chemical synthesis technology i.e. one compound is reacted with another in a chemical reaction in order to obtain a reaction product. Although these methods can be used in connection with the present invention, other recent technology involves the biological synthesis of peptides in large numbers. More specifically, the genetic material of bacteria or phage can be modified so that each bacterium or phage produce an individual peptide on their surface. By randomly producing large numbers of different pieces of altered genetic material it is possible to produce a mixture of bacteria or phage wherein the different bacteria or phage in the mixture include a different peptide expressed on the surface. One method of producing peptides on phage is taught by Devlin et al., Science (1990) 249:404-406 which discloses a method for the production and rapid evaluation of random libraries of millions of peptides on the surface of phage. A similar method was published by Scott and Smith, Science (1990) 249: 386-390. Scott and Smith disclose a method wherein peptides are produced on the surface of bacteriophage and the phage expressing a particular peptide tag can be selected from a mixture of tens of millions of clones expressing oligopeptides of random sequences using affinity purification with a protein ligand. Christian et al., J. Mol. Biol., (1992) 227:711-718 discloses simplified methods for the construction, assessment and rapid screening of peptide libraries in bacteriophage. Another related method involves the generation of libraries by insertion of peptides into the external domain of bacterial outer-membrane proteins, such as lam B, using recombinant technology Brown, Proc. Natl. Acad. Sci. USA (1992) 89: 8651-8655. Still another method of producing large numbers of peptides is taught in U.S. Pat. No. 5,223,409 issued Jun. 29, 1993 to Ladner et al.
All the above discussed methods for producing libraries of peptides and modified peptides can be used in connection with the present invention. Although the methods are extremely useful for producing large mixtures of peptides and modified peptides, the methodologies do not allow one to identify molecules which bind to antibodies in sera when neither the antibody nor the antigen is known, or to identify antibodies specific to a given disease. The present invention endeavors to provide technology which makes such possible.